The field of biomolecule screening for biologically and therapeutically relevant compounds is rapidly growing. Relevant biomolecules that have been the focus of such screening include chemical libraries, nucleic acid libraries and peptide libraries, in search of molecules that either inhibit or augment the biological activity of identified target molecules. With particular regard to peptide libraries, the isolation of peptide inhibitors of targets and the identification of formal binding partners of targets has been a key focus. However, one particular problem with peptide libraries is the difficulty assessing whether any particular peptide has been expressed, and at what level, prior to determining whether the peptide has a biological effect.
Green fluorescent protein (GFP) is a 238 amino acid protein. The crystal structure of the protein and of several point mutants has been solved (Ormo et al., Science 273, 1392-5, 1996; Yang et al., Nature Biotechnol. 14, 1246-51, 1996). The fluorophore, consisting of a modified tripeptide, is buried inside a relatively rigid beta-can structure, where it is almost completely protected from solvent access. The fluorescence of this protein is sensitive to a number of point mutations (Phillips, G. N., Curr. Opin. Struct. Biol. 7, 821-27,1997). The fluorescence appears to be a sensitive indication of the preservation of the native structure of the protein, since any disruption of the structure allowing solvent access to the fluorophoric tripeptide will quench the fluorescence.
Abedi et al (Nucleic Acids Res. 26, 623-30, 1998) have inserted peptides between residues contained in several GFP loops. Inserts of the short sequence LEEFGS (SEQ ID NO: 1) between adjacent residues at 10 internal insertion sites were tried. Of these, inserts at three sites, between residues 157-158, 172-173 and 194-195 gave fluorescence of at least 1% of that of wild type GFP. Only inserts between residues 157-158 and 172-173 had fluorescence of at least 10% of wild type GFP. When -SAG-random 20mer-GAS- peptide sequences were inserted at different sites internal to GFP, only two sites gave mean fluorescence intensities of 2% or more of the GFP-random peptide sequences 10-fold above background fluorescence. These sites were insertions between residues 157-158 and 172-173.
It is an object of the invention to provide compositions of fusion constructs of peptides with fluorescent proteins such as GFP, and methods of using such constructs in screening of peptide libraries.